This project is designed to define the morphologic, molecular, and metabolic characteristics of breast ducts and ductal epithelial cells at normal risk and increased risk for breast cancer. This data is needed to define the early changes in the carcinogenic pathway for breast cancer, to develop an improved classification and molecular signature of preneoplastic breast tissue for risk assessment, to identify new targets, and to facilitate selection and monitoring of women for breast cancer prevention. This project includes the following clinical and laboratory studies: a.) Protocol 02-C-0077, Characterization of High Risk Breast Duct Epithelium by Cytology, Breast Duct Endoscopy, and Gene Expression Profile (DN Danforth, PI). b.) Protocol 02-C-0144, Establishment of Normal Breast Epithelial Cell Cultures, and a High Risk Cell Line and Tissue Repository from Breast Tissue from Women at High Risk for Breast Cancer (DN Danforth, PI). c.) Comprehensive literature review of molecular changes in normal breast tissue at normal risk and at high risk for breast cancer, in hyperplasia, and in atypical hyperplasia to define the presence and sequence of accumulation of molecular changes in early breast carcinogenesis. d.) Regulation of proliferation and DNA damage repair in breast epithelium by endogenous risk factors. Protocol 02-C-0077 examines and characterizes by ductal lavage and ductal endoscopy the ducts and ductal epithelium of women at normal risk and increased risk for breast cancer. Seventy-seven women have been studied, 51 high risk subjects and 26 subjects at normal risk. Cytologic atypia of ductal epithelial cells was identified and correlated with ductal endoscopic findings of architectural changes. A new method for acquiring ductal epithelial cellular samples for cytologic and molecular analysis was developed: ductal endoscopic targeted sampling with brush sampling devices of normal epithelium. This provided significantly increased cellular yields and samples of greater than 90% pure ductal epithelial cells. A novel method of whole genome amplification of DNA from cytopathologic slides was developed, and molecular characterization of high risk breast epithelium is in progress. Initial studies will define the presence and incidence of hemizygous and homozygous deletions of the p16 and p53 genes in ductal breast epithelial cells using fine tiling CGH array of chromosome 9 and chromosome 17, with allele loss validated by Taqman p53 copy number assays. Downstream consequences of p16 and p53 loss, including telomere shortening and DNA methylation will be studied. In this manner a model will be developed for analyzing and characterizing allele loss of tumor suppressor genes in breast epithelium in early breast carcinogenesis. Breast ductal fluid and ductal epithelial cells are being studied for the presence of miRNA, noncoding transcripts which bind to mRNA and result in gene silencing. miRNA has been identified in exosomes of breast ductal fluid, suggesting an important mechanism for the expansion of the cancerized field within the breast and enhancement of progression through the carcinogenic pathway (Danforth DN et al. J Surg Oncol, 94:555-564, 2006). Protocol, 02-C-0144 establishes a tissue and cell line repository from all major sites of normal breast tissue at increased risk for breast cancer, including the contralateral normal breast, tissue adjacent to a breast cancer, women with a strong family history of breast cancer (including BRCA1 and BRCA2 mutation carriers), or a Gail model risk estimate of breast cancer of greater than 1.67%. Tissues are processed to allow for a wide spectrum of molecular studies. Mortal epithelial, fibroblast, and adipose cell lines are developed from normal breast tissue to allow for a wide range of phenotypic, metabolic, and molecular studies. Demographic data is collected for each subject, and all specimens are stored in an NIH repository. This protocol also represents an important source of fresh breast tissue for mammary stem cell analysis under the Breast Stem Cell Stamp Fund Project. A comprehensive literature review of the major molecular changes in preneoplastic breast tissue has been conducted to define the early phases of the breast cancer carcinogenic pathway and aid in the molecular analysis of normal risk and high risk breast tissue. Four types of preneoplastic breast tissue - normal/benign breast tissue at low risk for breast cancer, normal/benign tissue at high risk, epithelial hyperplasia, and epithelial hyperplasia with atypia - were reviewed to define a.) numerical chromosomal changes, including partial and whole chromosome gain or loss;b.) structural chromosome changes, including allelic imbalance, microsatellite instability, loss of heterozygosity, amplifications, and chromosomal rearrangements;and c.) patterns and classification of protein and gene expression changes, including epigenetic changes, oncogene amplifications, tumor suppressor gene loss, and cell cycle gene alterations. These patterns were studied both individually and globally across the four types of preneoplastic breast tissue. Molecular changes were correlated with risk for breast cancer and with progression between histologic/morphologic subtypes. This review indicated that the earliest molecular changes in breast carcinogenesis are loss of heterozygosity and DNA methylation of tumor suppressor loci, present in morphologically normal low risk and normal high risk breast tissue, while aneusomy is a later event and is first identified in high risk normal breast tissue. Gene amplification is an uncommon early event in preneoplastic tissues. Molecular changes accumulate with progression through the carcinogenic pathway from normal breast tissue to hyperplasia and atypia. This characterization provides an important model for guiding molecular analysis and defining the carcinogenic pathway in preneoplastic breast tissue. Proliferation of normal breast epithelial cells at increased risk for breast cancer is a critical determinant for clonal expansion and for the accumulation of genetic abnormalities in breast carcinogenesis. To understand regulation of this proliferation, the effects of two prominent stimulatory and risk factors for breast cancer, estradiol (E2) and insulin-like growth factor-1 (IGF-1) on normal and high risk breast epithelial cells were studied. IGF-1 stimulated growth of all breast epithelial cells in a time-dependent and dose-dependent manner without modulation of apoptosis. This proliferative action by IGF-1 was accompanied by the rapid stimulation of phosphorylation of IGF-1R and IRS-1, and by downregulation of IRS-1 at the posttranscriptional level (protein expression) and of IRS-2 at the transcriptional level (gene and protein expression). These cells express estrogen receptor alpha (ERalpha) and beta (ERbeta) and progesterone (PR) receptors;however, estradiol did not stimulate proliferation or cell cycle progression and did not modulate ER or PR in any of these cells. Importantly, IGF-1 acted synergistically with estradiol to stimulate growth in a high risk breast epithelial cell line (MCF12A) but not in any normal risk cell lines (MCF10A, AG11132, AG11134), suggesting that the transition to estradiol responsiveness and synergism with IGF-1 may occur at or beyond the level of hyperplasia in the carcinogenic pathway. Further, these findings indicate that IGF-1 is the dominant mitogen in early breast carcinogenesis, and estrogen responsiveness of normal breast epithelial cells and modulation of IGF-1 signaling occur later in the carcinogenic pathway. The role of IGF-1 in enhancing chromosomal instability in early carcinogenesis will be defined by studying its modulation of DNA damage repair and double-strand breaks and signaling in normal and increased risk breast epithelium.